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Automated synthesis of S. pneumoniae 7F capsular polysaccharide repeating unit as candidate for conjugate vaccines

Periodic Reporting for period 1 - AUTOOLIGO (Automated synthesis of S. pneumoniae 7F capsular polysaccharide repeating unit as candidate for conjugate vaccines)

Reporting period: 2015-04-01 to 2017-03-31

Pneumococcal infections cause millions of deaths among children and the elderly worldwide, despite the widespread use of a blockbuster glycoconjugate vaccine Prevnar13®.[1] This vaccine induces an immune response against bacterial cell-wall glycans which are not present on human cells.[2] Although it has dramatically reduced mortality caused by invasive pneumococcal diseases (IPD), several issues prevail, such as insufficient serotype coverage, troublesome purification from isolated capsular polysaccharides (CPSs) and lack of efficacy against certain serotypes.[2] Chemically defined synthetic oligosaccharides containing the protective glycan epitope (glycotope) are an attractive alternative to isolated CPSs. They induce immune response to the native CPSs and avoid problems common for CPSs, such as contamination with pneumococcal cell wall polysaccharide, various chain lengths and multiple coupling sites.

The CPS of S. pneumoniae serotype 3 (ST3) has a disaccharide repeating unit (1) consisting of β-linked glucuronic acid and glucose (cellobiuronic acid) (Fig. 1). It is known to cause invasive pneumococcal infections both in children and adults. Although it is covered by highly successful pneumococcal conjugate vaccine Prevnar13®, it has limited efficacy and remains the main cause of invasive pneumococcal disease in Europe.[3] Recently, a tetrasaccharide 2-CRM197 conjugate has been identified as a synthetic vaccine candidate.[4] In order to gain deeper understanding of the role of the terminal sugar as well as to study antibody binding, two trisaccharides 3 and 4 representing frameshifts of the repeating unit were designed and prepared (Fig. 1).

Streptococcus pneumoniae serotype 7F (ST7F) is the second major cause of IPD in Europe, infecting mainly adult population.[3] Its repeating unit (RU) structure was assigned in 1988.[6] The branched heptasaccharide ST7F CPS RU 5 is composed of a linear sequence of D-galactose (D-Gal), 2-O-acetyl-L-rhamnose (L-RhaOAc), D-glucose (D-Glc) and N-acetyl-D-galactoseamine (D-GalNAc). A two-sugar side chain branching from D-GalNAc contains L-rhamnose (L-Rha) and N-acetyl-D-glucoseamine (D-GlcNAc). A second branch leads from D-Gal and is formed by another D-Gal unit (Fig 1). In 1992, Kamerling et al. reported the synthesis of O-deacetylated L-Rha-D-Glc-D-GalNAc trisaccharide.[7] To the best of our knowledge, there are no other reports concerning the synthesis or immunology of ST7F oligosaccharide antigens.

In order to identify the minimal glycotope that can elicit a robust immune response to the CPS, we designed a series of oligosaccharides related to the RU of ST7F CPS. The effect of branching, length and the role of deacetylation on overall immunogenicity was evaluated. Each compound was equipped with a reducing-end C5 linker to enable printing on glycan arrays and conjugation to a carrier protein.

[1] Nat. Rev. Micro, 2009, 7, 838-838.
[2] B. Schumann, C. Anish, C. L. Pereira and P. H. Seeberger, in Biotherapeutics: Recent Developments using Chemical and Molecular Biology, The Royal Society of Chemistry, 2013, DOI: 10.1039/9781849737159-00068 pp. 68-104.
[3] ECDC Report, 2014.
[4] S. G. Parameswarappa, K. Reppe, A. Geissner, P. Ménová, S. Govindan, Adam D. J. Calow, A. Wahlbrink, Markus W. Weishaupt, Bopanna P. Monnanda, Roland L. Bell, L.-A. Pirofski, N. Suttorp, Leif E. Sander, M. Witzenrath, Claney L. Pereira, C. Anish and Peter H. Seeberger, Cell Chem. Biol., 2016, 23, 1407-1416.
[5] X. Wu, C.-C. Ling and D. R. Bundle, Organic Letters, 2004, 6, 4407-4410.
[6] M. Moreau, J. C. Richards, M. B. Perry and P. J. Kniskern, Carb. Res., 1988, 182, 79-99.
[7] A. M. P. van Steijn, J. P. Kamerling and J. F. G. Vliegenthart, J. Carb. Chem., 1992, 11, 665-689.
Project 1: Synthesis of trisaccharide motives from CPS of Streptococcus pneumoniae serotype 3
Two different protecting-group strategies have been applied in the synthesis of the target trisaccharides: the first based on TMS and the other on 2-naphthylmethyl as temporary protecting groups. The desired trisaccharides have been successfully prepared using both strategies. We have published the first strategy [4] and filed a patent on the other.

The prepared trisaccharides were conjugated to diphtheria detoxified toxin CRM197, a commonly used carrier protein in licensed vaccines, using p-nitrophenyl adipate ester as coupling agent.[5] The conjugates were co-formulated with an adjuvant (aluminium hydroxide or Freund’s) and used to immunize the mice. The immune response was assessed using ELISA and glycan arrays. It was proved that both constructs are immunogenic and produced antibodies that differ depending on which trisaccharide was used for the immunization. Further experiments (e.g. opsonophagocytic killing assay and agglutination assay) will be done to characterize these antibodies.

Project 2: Synthesis of oligosaccharides related to CPS repeating unit of Streptococcus pneumoniae serotype 7F
The synthesis started with the procurement of the differentially protected monosaccharide building blocks by established literature procedures. A series of glycosylation and deprotection steps provided fully protected oligosaccharides. Removal of all protective groups furnished four target compounds.

Glycan arrays containing the prepared glycans were used to map the glycotopes that are recognized by antibodies raised against natural CPS. The four prepared oligosaccharides as well as native ST7F CPS and related structures from other S. pneumoniae strains were covalently immobilized on glass slides and probed with antibodies. The resulting glycan array was probed with rabbit anti-ST7 serum, a polyclonal serum that is specifically raised by immunizing animals with isolated ST7 CPS. The glycan array revealed specific binding with two synthetic glycans. Further experiments are necessary to prove our hypothesis about the protective epitope.

Dissemination and public engagement
- TMS strategy published in [4]
- manuscript on Nap strategy in preparation
- manuscript on ST7F in preparation

- Improved preparation of vaccines against Streptococcus pneumoniae type 3 - filed
- Another patent on ST3 currently with patent lawyers

The results from the ST3 project were presented at two conferences (IBS Melbourne and ICS New Orleans) and during an invited talk at ICT Prague. Additionally, 3 general talks on carbohydrates and vaccines were held for high-school students.
Project 1: Synthesis of trisaccharide motives from CPS of Streptococcus pneumoniae serotype 3
- two trisaccharide structures synthesized using new protecting-group strategies
- ST3 tetrasaccharide currently taken to clinical trials

Project 2: Synthesis of oligosaccharides related to CPS repeating unit of Streptococcus pneumoniae serotype 7F
- four new structures related to ST7F repeating unit synthesized and evaluated for immunogenicity

A spin-off company Vaxxilon has been founded to take over the development of semi-synthetic glycoconjugate vaccines against S. pneumoniae. Serotype 3 glycoconjugate vaccine has just been approved for clinical trials.
Figure 3
Figure 2
Figure 1